A limited number of proteins have been identified as being primarily associated with bone tissue. In addition, reports in the literature have presented compelling evidence that the targets of these proteins are receptors on the cell surface. Protein domains may be responsible for targeting these proteins to cell receptors or receptor ligands in the extracellular matrix of the target tissue. Therefore, these peptides could be used for a wide variety of therapeutic uses including the delivery of drugs that could help in the treatment of musculoskeletal disorders, genetic or acquired, osteoporosis and metastatic cancer. Furthermore, it is anticipated that the peptides themselves can serve as therapeutic agents providing osteogenic or tropic activity for osteoblast, mesenchymal or hematopoietic cell lineages.
Although much progress has been made in the field of bone regenerative medicine during the past few years, current therapies that use bone grafts still have their limitations. In spite of the fact that material science technology has lead to clear improvements in the field of bone repair, no adequate bone substitute has been developed and hence injuries that produce large bone defects still represent a major challenge for orthopedic and reconstructive surgeons. Delivery of osteoinductive factors, such as bone morphogenetic proteins (BMPs), has been successfully applied to augment local bone repair and several formulations are available for clinical applications. However, the wide-spread clinical efficacy of these treatments continues to be hampered by inadequate delivery of carriers, release kinetics, dosage and potency.
It is clear that an adequate bone replacement is yet to be found and that it is needed for full recovery of large bone defect. Phage display peptide libraries have enabled the discovery of peptides that selectively target specific organs. Much progress has been made in this rapidly developing field and many possible applications of phage technology have been developed. These include the creation and screening of libraries to discover novel therapeutic targets and methods for selection of biologically active ligands.
Approximately 5% to 10% of fractures sustained in the United States are associated with delayed healing or nonunion. Impaired fracture-healing is associated with a number of risk factors, including poor blood supply, associated soft-tissue injury, extensive bone loss, instability, infection, poor general medical condition, and unhealthy habits, such as smoking. Traditionally, problems related to fracture-healing have been treated with operative intervention, which often involves the use of an autogenous bone graft. However, bone graft-harvesting procedures are associated with a morbidity rate of 10% to 30%, and only limited amounts of autogenous bone are available. Allograft provides an osteoconductive scaffold but lacks osteoinductive properties. There is also a concern about possible disease transmission. Therefore, alternative strategies designed to enhance the healing of acute fractures and to improve the treatment of delayed unions and nonunions are required.
A number of growth factors are expressed during different phases of experimental fracture-healing. Both in vitro and in vivo studies have shown that growth factors, such as transforming growth factor beta (TGF-β), bone morphogenetic protein (NMP), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF), have varying amounts of osteoinductive potential. Among these growth factors, BMPs appear to have the most osteoinductive potential. The BMPs are members of the TGF-β superfamily, and more than a dozen different molecules have been identified at this time. Presently, BMP-2, 4, and 7 are known to play a critical role in bone-healing by virtue of their ability to stimulate differentiation of mesenchymal cells to an osteochondroblastic lineage.
It is believed that synthetic peptides have several advantages over large molecular growth factor proteins: (1) they can be synthesized on a large scale at a relatively low cost and with little variation between different batches; (2) they are more stable than large-molecule growth factors and easier to handle; (3) they have long shelf lives, both as free and as immobilized peptides, and do not require special handling and storage procedures or facilities; (4) the peptides and their biomaterial composites can be sterilized easily by autoclaving, by UV, or by gamma-irradiation; and (5) they do not induce inflammatory responses in vivo.
Biologically, the peptides may have other advantages, among the most relevant of which is a broad potential for bioengineering applications. They could be used to reduce the effective dose of growth factors within a medical device and could be of particular use for coating onto device surfaces. They could also maximize the biological activity of biological preparations such as demineralized bone matrix (DMB). Furthermore, they peptides might augment the endogenous levels of growth factors generated by host tissue during bone healing. The discovery of osteogenic peptides therefore may open up a wide variety of practical applications for the treatment of fracture non-unions or of bone defects.
Recently, the osteogenic growth peptide (OGP) and its analogues, such as OGP-14, have attracted considerable clinical interest. OGP is a naturally occurring tetradecapeptide identical to the C-teiniinal amino acid sequence comprising residues 89-102 of histone H4. OGP and its analogues increase bone formation and trabecular bone density and stimulate fracture healing when administered to mice and rats. In cultures of osteoblastic and other bone marrow stromal cells derived from human and other mammalian species, OGP regulates proliferation, alkaline phosphatase activity, and matrix mineralization. The thrombin-related peptide, TP508, is a 23 amino acid peptide representing the natural amino acid sequence of the receptor-binding domain of human thrombin. P-15 is a 15-residue synthetic analogue of a cell-binding domain of type I collagen. Both of these peptides have the ability to enhance bone regeneration in rodents, however, they are not bone targeting peptides and do not modify osteogenesis directly. TP508 promotes bone regeneration by stimulating revascularization of granulation tissue at the injured site, while P15 mimics the cell binding domain of type I collagen, which is an extracellular matrix protein with multiple cell-binding domains for osteogenic progenitor cells, to play a role in osteogenesis.
Random peptide phage display libraries have been used in vivo with nonrandom distribution of peptides isolated from phage bound to different organs. This specificity of phage binding as imparted by the peptide epitope has allowed investigators to determine that each organ's microvasculature had unique determinants. The process of in vivo phage display has led to the identification of interacting receptors on cells. In addition to the cell surface targets that were found for phage display peptides, there have been reports of an intracellular fate for phage display vectors in mammalian cells.
These observations have raised the possibility that peptides identified by in vivo biopanning of a phage display library may be useful for targeted delivery of genes for therapeutic purposes and perhaps for their delivery to cells in specific organs. Furthermore, peptides that are directed to cell surface components, such as CD34 may prove useful for cell selection. Screening the phage library can lead to the identification of ligands to cell surface proteins in target cells and tissues. Since the construction of the peptide phage display library is random by design, many of the peptides that interact specifically with any given target tissue may not represent true protein epitopes but are likely to be protein mimotopes. These sequences from phage display libraries may be helpful as structural and functional mimics that could serve as the basis for novel drug design for the interacting target. In vivo biopanning has been used to identify peptides that exhibit organ-specific homing for the kidney and brain and later for other organs and tumor vasculature in animals.
There is a long felt need in the art to identify peptides useful to treat diseases and disorders related to abnormal cell adhesion, angiogenesis, and osteogenesis. The present invention satisfies these needs.